Method for the enzymatic production of 2-hydroxy-2-methyl carboxylic acids

ABSTRACT

The present invention relates to a method for the enzymatic production of 2-hydroxy-2-methyl carboxylic acids from 3-hydroxy carboxylic acids, where a 3-hydroxy carboxylic acid is produced in an aqueous reaction solution and/or is added to this reaction solution and is incubated. The aqueous reaction solution comprises a unit having 3-hydroxy-carboxylate-CoA mutase activity which has both 3-hydroxy-carbonyl-CoA ester-producing and 3-hydroxy-carbonyl-CoA ester-isomerizing activity and converts the 3-hydroxy carboxylic acid into the corresponding 2-hydroxy-2-methyl carboxylic acid which is isolated as acid or in the form of its salts. In a preferred embodiment, the unit having 3-hydroxy-carboxylate-CoA mutase activity is a unit which includes an isolated cobalamin-dependent mutase and where appropriate a 3-hydroxy-carbonyl-CoA ester-producing enzyme or enzyme system or a microorganism including them. The invention preferably relates to a biotechnological process for producing 2-hydroxy-2-methyl carboxylic acids, where microorganisms which have the desired activities are cultured in an aqueous system with the aid of simple natural products and convert intracellularly formed 3-hydroxy-carbonyl-CoA esters into the corresponding 2-hydroxy-2-methyl carboxylic acids. The invention likewise encompasses the production of unsaturated 2-methyl carboxylic acids, where the 2-hydroxy-2-methyl carboxylic acids obtained are converted by dehydration into the corresponding unsaturated 2-methyl carboxylic acids (methacrylic acid and higher homologues).

The present invention relates to a method for the enzymatic productionof 2-hydroxy-2-methyl carboxylic acids from 3-hydroxy carboxylic acids,where a 3-hydroxy carboxylic acid is produced in an aqueous reactionsolution and/or is added to this reaction solution and is incubated. Theaqueous reaction solution comprises a unit having3-hydroxy-carboxylate-CoA mutase activity which has both3-hydroxy-carbonyl-CoA ester-producing and 3-hydroxy-carbonyl-CoAester-isomerizing activity and converts the 3-hydroxy carboxylic acidinto the corresponding 2-hydroxy-2-methyl carboxylic acid which isisolated as acid or in the form of its salts. In a preferred embodiment,the unit having 3-hydroxy-carboxylate-CoA mutase activity comprises anisolated cobalamin-dependent mutase and where appropriate a3-hydroxy-carbonyl-CoA ester-producing enzyme or enzyme system or is amicroorganism including them. The invention preferably relates to abiotechnological process for producing 2-hydroxy-2-methyl carboxylicacids, where microorganisms which have the desired mutase activity arecultured in an aqueous system with the aid of simple natural productsand intracellularly formed 3-hydroxy-carbonyl-CoA esters are convertedinto the corresponding 2-hydroxy-2-methyl carboxylic acids. Theinvention likewise encompasses the production of unsaturated 2-methylcarboxylic acids, where the 2-hydroxy-2-methyl carboxylic acids obtainedare converted by dehydration into the corresponding unsaturated 2-methylcarboxylic acids (methacrylic acid and higher homologs).

In a preferred embodiment of the invention, the 3-hydroxy-carbonyl-CoAthioester-producing and 3-hydroxy-carbonyl-CoA thioester-isomerizingmicroorganism used is the strain HCM-10 (DSM 18028).

Methacrylic acid and homologous unsaturated 2-methyl carboxylic acidsare widely used in the production of acrylic glass sheets,injection-molded products, coatings and many other products.

A plurality of processes for the production of methacrylic acid and itshomologs have been disclosed. However, the vast majority of thecommercial production worldwide is based on a method of hydrolyzing theamide sulfates of methacrylic acid and its homologs, which are producedfrom the corresponding 2-hydroxy nitrites (W. Bauer, “Metharylic acidand derivatives”, in: Ullmann's Encyclopedia of Industrial Chemistry,5th edition, editors: B. Elvers, S. Hawkins, G. Schulz, VCH, New York,1990, Vol. A16, pp. 441-452; A. W. Gross, J. C. Dobson, “Methacrylicacid and derivatives”, in Kirk-Othmer Encyclopedia of ChemicalTechnology, 4th edition, editors: J. I. Kroschwitz, M. Howe-Grant, JohnWiley & Sons, New York, 1995, Vol. 16, pp. 474-506). This methodrequires, for example, about 1.6 kg of sulfuric acid for the productionof 1 kg of methacrylic acid. For this reason, alternative methods forthe commercial production of methacrylic acid without the requirement ofrecovering the sulfuric acid (and the high energy costs associatedtherewith) would be advantageous.

U.S. Pat. No. 3,666,805 and U.S. Pat. No. 5,225,594 have disclosed thechemical conversion of 2-hydroxy isobutyric acid to methacrylic acid.This comprises dehydrating 2-hydroxy isobutyric acid by using metaloxides, metal hydroxides, ion exchange resins, alumina, silicon dioxide,amines, phosphines, alkali metal alkoxides and alkali metalcarboxylates. Usual reaction temperatures are between 160° C. and 250°C. This method made possible methacrylic acid yields of up to 96%.

An alternative method for the production of methacrylic acid and itshomologs is based on the hydrolysis of 2-hydroxy nitrites to thecorresponding 2-hydroxy-2-methyl carboxylic acids, utilizingnitrile-hydrolyzing enzymes. The latter are nitrilase or a combinationof nitrile hydratase and amidase (A. Banerjee, R. Sharma, U. C.Banerjee, 2002, “The nitrile-degrading enzymes: current status andfuture prospects”, Appl. Microbiol. Biotechnol., 60:33-44). This methodis protected by a plurality of patents (U.S. Pat. No. 6,582,943 B1). Asevere disadvantage of this method is the instability of the nitrites inthe neutral pH range required for an efficient nitrile-hydrolyzingenzyme activity. The decomposition of the nitriles in the reactionmixture results in accumulation of ketones and cyanides, both of whichinhibit the nitrile-hydrolyzing enzyme activities.

A general disadvantage of both methods, i.e. of the currently dominatingmethod based on amide sulfates and of the enzymatic nitrile-hydrolyzingmethod, is the need for 2-hydroxy nitrites. The latter must first beprepared from environmentally harmful reactants, namely ketones andcyanide.

For this reason, methods for the production of methacrylic acid and itshomologs, which are based on simple environmentally benign reactants,would be advantageous.

It was therefore the object of the invention to search for alternativepossibilities of producing 2-hydroxy-2-methyl carboxylic acids and toprovide methods which are based, where possible, on the application ofsimple, environmentally benign reactants, consume little energy andproduce few waste products.

The object is achieved by an enzymatic method for the production of2-hydroxy-2-methyl carboxylic acids from 3-hydroxy carboxylic acids.According to the invention, said 3-hydroxy carboxylic acid is producedin and/or added to an aqueous reaction solution which has a unit having3-hydroxy-carboxylate-CoA mutase activity. A unit having3-hydroxy-carboxylate-CoA mutase activity means for the purpose of theinvention a unit comprising a cobalamin-dependent mutase and, whereappropriate, a 3-hydroxy-carbonyl-CoA ester-producing enzyme or enzymesystem or a biological system comprising or producing them, which have3-hydroxy-carboxylate-CoA mutase activity and exhibit both3-hydroxy-carbonyl-CoA ester-producing and 3-hydroxy-carbonyl-CoAester-isomerizing activity. After incubation the correspondinglyconverted 2-hydroxy-2-methyl carboxylic acid is then isolated as acid orin the form of its salts.

The invention preferably relates to a biotechnological process for theproduction of 2-hydroxy-2-methyl carboxylic acids with the use ofmicroorganisms. Said microorganisms usually have 3-hydroxy-carbonyl-CoAester-synthesizing activity and are capable of producing or comprisesuch a cobalamin-dependent mutase and, due to the3-hydroxy-carboxylate-CoA mutase activity, are capable of convertingintracellularly 3-hydroxy-carbonyl-CoA esters formed from simple naturalproducts (from reactants such as, for example, sugars and/or alcoholsand/or organic acids and their derivatives) to the corresponding2-hydroxy-2-methyl-carbonyl CoA esters.

The method of the invention is characterized in particular in thatmicroorganisms which produce or comprise the cobalamin-dependent mutaseand have 3-hydroxy-carboxylate-CoA mutase activity are used in aqueoussystems for converting 3-hydroxy carboxylic acids to the corresponding2-hydroxy-2-methyl carboxylic acid.

In a preferred variant method, microorganisms which comprise3-hydroxy-carboxylate-CoA mutase activity and have both3-hydroxy-carbonyl-CoA thioester-producing and 3-hydroxy-carbonyl-CoAthioester-isomerizing activity are cultured in an aqueous system withrenewable raw materials or waste products deriving from the consumptionof renewable raw materials as carbon and energy sources. In the process,the intracellularly formed 3-hydroxy-carboxylate-CoA thioesters areconverted to the corresponding 2-hydroxy-2-methyl carboxylic acids. Thereaction is preferably carried out with the addition of external3-hydroxy carboxylic acid. The corresponding 2-hydroxy-2-methylcarboxylic acid is then isolated as acid or in the form of its salts.

This novel biotechnology method which utilizes the production of3-hydroxy carboxylic acids from simple natural products and theirisomerization to 2-hydroxy-2-methyl carboxylic acids is capable ofsolving the problem specified above.

In a preferred embodiment of the invention, the method comprises thefollowing steps

-   (a) 3-hydroxy carboxylic acids are produced from simple natural    products and then converted to 2-hydroxy-2-methyl carboxylic acids    in a suitable biological system which has 3-hydroxy-carbonyl-CoA    ester-synthesizing activity and mutase activity, and-   (b) the 2-hydroxy-2-methyl carboxylic acids are isolated as free    acids or as their corresponding salts.

The 2-hydroxy-2-methyl carboxylic acids obtained in this way may be usedadvantageously for producing C2-C3-unsaturated iso-alkenoic acids(methacrylic acid and its homologs), possibly by dehydration of theacids produced in (a) and (b) or their corresponding salts. Thesereactions are depicted below:

-   -   simple natural products (e.g. renewable raw materials or waste        products deriving from the consumption of renewable raw        materials, such as, for example, sugars, organic acids or        alcohols)→3-hydroxy carboxylic acids→2-hydroxy-2-methyl        carboxylic acids (e.g. by strain HCM-10)    -   2-hydroxy-2-methyl carboxylic acids→methacrylic acid and        homologs (e.g. in the presence of NaOH and a temperature of 185°        C.).

The reaction conditions (pH, ion concentration, oxygen/carbon dioxiderequirements, trace elements, temperatures and the like) are of coursechosen here in such a way that the microorganisms are enabled tooptimally convert 3-hydroxy carboxylic acids to 2-hydroxy-2-methylcarboxylic acids. Under these process conditions, thecobalamin-dependent mutase may have higher stability and efficacy in thenatural micro environment, i.e. inside the cell, than the isolatedenzyme. In addition, cell propagation and thus an increase in mutaseconcentration may be possible under suitable conditions. The enzymaticconversion by means of microorganisms thus constitutes, whereappropriate, an important advantage regarding reliability, automationand simplicity as well as quality and yield of the final product of themethod.

For the enzymatic conversion according to the invention of 3-hydroxycarboxylic acids to 2-hydroxy-2-methyl carboxylic acids, it is alsopossible to introduce into the reaction solution the unit having3-hydroxy-carboxylate-CoA mutase activity, i.e. a cobalamin-dependentmutase, preferably in combination with a CoA ester-synthesizingactivity, in a purified, concentrated and/or isolated form, it beingpossible for the enzymes to be of natural origin, for example. Theenzymes may of course be recombinantly produced enzymes from agenetically modified organism.

For the purpose of the invention, the enzymes are used in the method ofthe invention as catalysts both in the form of intact microbial cellsand in the form of permeabilized microbial cells. Further possible usesare those in the form of components (one or more) from microbial cellextracts, but also in a partially purified or purified form. Whereappropriate, other CoA ester-synthesizing enzymes, for example CoAtransferase or CoA sythetases, are used according to the invention. Theenzymatic catalysts may be immobilized or attached to a dissolved orundissolved support material.

In a preferred variant embodiment, particular cell compartments or partsthereof separated from one another or combined, i.e. carbohydratestructures, lipids or proteins and/or peptides and also nucleic acids,which are capable of influencing the unit having mutase activity in apositive or negative way may be combined or separated. In order toutilize such an influence consciously, for example, crude extracts areprepared from the microorganisms in an expert manner for example, whichextracts are centrifuged, where appropriate, to be able to carry out areaction of the invention with the sediment or the supernatant.

3-hydroxy carboxylic acids (for example 3-hydroxy butyric acid) or morespecifically their intracellular CoA thioester 3-hydroxy-carbonyl-CoA,may readily be produced from simple natural products by a large numberof bacteria strains. These acids are the basic building blocks/monomersfor the common bacterial carbon and energy storage substance,poly-3-hydroxyalkanoate. Rearrangements of the carbon within theskeleton of carboxylic acids are likewise common in bacterial as well asin other biological systems. However, no biological system forconverting 3-hydroxy-carbonyl-CoA esters to the corresponding2-hydroxy-2-methyl-carbonyl-CoA esters has previously been identified.The invention is based on the surprising finding that systems withcobalamin-dependent mutase activity have both properties.

Microorganisms comprising cobalamin-dependent mutases are, for example,Methylibium petroleiphilum PM1, Methylibium sp. R8 (strain collectionUFZ, Leipzig, Germany), the β-proteobacterial strain HCM-10,Xanthobacter autotrophicus Py2, Rhodobacter sphaeroides (ATCC17029) orNocardioides sp. JS614.

A preferably suitable biological system has been found in the strainHCM-10. Said strain has been deposited in accordance with the BudapestTreaty on the deposit of microorganisms for the purposes of patentprocedure at the Deutschen Sammlung von Microorganismen und ZellkulturenGmbH [German collection of microorganisms and cell cultures], Brunswick,Germany, under No. DSM 18028 on 13.03.2006.

Using this preferred biological system, it has been possible to achievea particularly good yield of 2-hydroxy-2-methyl carboxylic acids, inparticular 2-hydroxy isobutyric acid. However, the enzymatic conversionby microorganisms is not at all limited to this strain. Any organismscapable of converting 3-hydroxy carboxylic acids to 2-hydroxy-2-methylcarboxylic acids may be used according to the invention.

They may be microorganisms which firstly possess the same gene or geneproduct or secondly have an analogous gene resulting in gene productshaving a similar or analogous activity. I.e., 3-hydroxy-carbonyl-CoAmutase activities of other origin are likewise covered by the invention.The invention also includes transformed systems which have a or asimilar 3-hydroxy-carbonyl-CoA mutase activity as strain HCM-10 or thatof other origin.

This may include mutants, genetically modified and isolatedmodifications of the microorganisms, for example organisms which havethe desired cobalamin-dependent mutase activity owing to theintroduction of a mutase-encoding nucleotide sequence.

The preferably used biological system (strain HCM-10-DSM 18028) produces3-hydroxy-carbonyl-CoA esters as thioesters from simple natural productssuch as sugars and/or organic acids and/or alcohols and theirderivatives. In the preferred system used herein, the3-hydroxy-carbonyl-CoA esters are converted by the cobalamin-dependentcarbon skeleton-rearranging mutase to 2-hydroxy-2-methyl-carbonyl-CoAesters, as depicted by way of example for the case of(R)-3-hydroxy-butyryl CoA in equation 1. The CoA thioester is hydrolyzedin the system and the acid is secreted into the culture medium.

Preference is given to using as enzyme catalysts in the method of theinvention the microorganism strains comprising cobalamin-dependentmutases, HCM-10 (DSM 18028), Xanthobacter autotrophicus Py2, Rhodobactersphaeroides (ATCC17029) or Nocardioides sp. JS614, their crude extractsor parts. The strains used according to the invention preferably producethe proteins with the sequences SEQ ID NO: 2 and/or SEQ ID NO: 4 orcomprise the nucleic acid sequences SEQ ID NO: 1 and/or SEQ ID NO: 3(HCM-10), the proteins with the sequences SEQ ID NO: 5 and/or SEQ ID NO:6, or comprise the nucleic acid sequences SEQ ID NO: 7 and/or SEQ ID NO:8 (Xanthobacter autotrophicus Py2), the proteins with the sequences SEQID NO: 9 and/or SEQ ID NO: 10, or comprise the nucleic acid sequencesSEQ ID NO: 11 and/or SEQ ID NO: 12 (Rhodobacter sphaeroides ATCC 17029)or the proteins with the sequences SEQ ID NO: 13 and/or SEQ ID NO: 14,or comprise the nucleic acid sequences SEQ ID NO: 15 and/or SEQ ID NO:16 (Nocardioides sp. JS614). For the purposes of the invention, saidproteins may also be used in a concentrated, isolated or syntheticallyproduced form.

In a further preferred variant embodiment of the invention, the enzymecatalysts, in particular microorganisms, crude extracts, parts thereofand/or the concentrated or isolated enzymes are used in an immobilizedform. Immobilization renders enzymes, cell organelles and cellsinsoluble and limited in reaction space. For example, they may beimmobilized in a polymer matrix (e.g. alginate, polyvinyl alcohol orpolyacrylamide gels). They may also be immobilized on dissolved orundissolved support materials (e.g. celite) to facilitate catalystrecovery and reuse. Methods of cell immobilization in a polymer matrixor on a dissolved or undissolved support are known to the skilled workerand have been described in detail previously. The enzyme activities maylikewise be isolated from the microbial cells. They may then be useddirectly as catalyst or in an immobilized form in a polymer matrix or ona dissolved or undissolved support. The methods required for this areknown to the skilled worker and, for example, described in Methods inBiotechnology, Vol. 1: Immobilization of enzymes and cells, editor: G.F. Bickerstaff, Humana Press, Totowa, N.J., 1997.

3-hydroxy carboxylic acids are converted to 2-hydroxy-2-methylcarboxylic acids preferably within the framework of a continuous processwhich may be carried out in a flow reactor in which microbial growth andthus product formation takes place. However, a continuous process mayalso mean any system of growing cells and catalyzing enzymes, which issupplied with nutrient solution and from which culture solution,including enzymatically formed 2-hydroxy-2-methyl carboxylic acid isremoved. According to the invention, the process may also be carried outas semicontinuous or batch process.

As explained above, 3-hydroxy carboxylic acid which is the startingmaterial for 2-hydroxy-2-methyl carboxylic acid is produced preferablyby enzymatic conversion of carbohydrates and/or organic acids and/oralcohols or their derivatives. In the context of the invention, use ismade, aside from the cobalamin-dependent mutase, where appropriatefurthermore of CoA ester-synthesizing enzymes which are present in oradded to the microorganism. This involves the conversion of hydrocarbonsand/or carbohydrates and/or organic acids and/or alcohols or derivativesthereof to the 3-hydroxy carboxylic acid and of the 3-hydroxy carboxylicacid to the 2-hydroxy-2-methyl carboxylic acid in a single process step,i.e. conversion of the starting substrates up to 3-hydroxy carboxylicacid and the enzymatic conversion reactions of 3-hydroxy carboxylic acidto the corresponding 2-hydroxy-2-methyl carboxylic acid are carried outat the same time or with a slight time delay in one and the samereaction solution.

In a very particular embodiment of the invention, a substrate with atert-butyl radical as carbon source and energy source is used forculturing, with preference being given to tert-butyl alcohol being thesole carbon and energy source in a basal medium.

The method of the invention is preferably useful for the production of2-hydroxy-2-methyl propanoic acid (2-hydroxy isobutyric acid). Thepreferred production of 2-hydroxy isobutyric acid is furthermorecharacterized in that 3-hydroxy butyric acid is added externally.

The method may be carried out aerobically, preferably with the use ofintact cells, or else unaerobically, for example with gassing withnitrogen, preferably when extracts or purified enzymes are used.

The invention also relates to nucleic acid molecules coding for anenzyme having the activity of a cobalamin-dependent mutase, selectedfrom the group consisting of

-   a) nucleic acid molecules coding for a protein comprising the amino    acid sequences indicated under Seq. No. 2 and/or Seq. No. 4;-   b) nucleic acid molecules comprising the nucleotide sequence    depicted under Seq. No. 1 and/or Seq. No. 3.

An enzyme of the invention has been shown to be preferably aheterodimeric protein which comprises the sub units described under Seq.No. 2 and Seq. No. 4 and thus has excellent enzyme activity.

A nucleic acid molecule may be a DNA molecule, preferably cDNA orgenomic DNA and/or an RNA molecule. Both nucleic acids and proteins maybe isolated from natural sources, preferably from DSM 18028, but also,for example, from Methylibium petroleiphilum PM1, Methylibium sp. R8(strain collection UFZ Leipzig, Germany), Xanthobacter autotrophicusPy2, Rhodobacter sphaeroides (ATCC17029) or Nocardioides sp. JS614 orthey may be synthesized by known methods.

Mutations may be generated in the nucleic acid molecules used accordingto the invention by means of molecular biology techniques known per se,thereby enabling further enzymes with analogous or similar properties tobe synthesized, which are likewise used in the method of the invention.Mutations may be deletion mutations which result in truncated enzymes.Modified enzymes with similar or analogous properties may likewise begenerated by other molecular mechanisms such as, for example,insertions, duplications, transpositions, gene fusion, nucleotideexchange or else gene transfer between different microorganism strains.

Such nucleic acid molecules may be identified and isolated using thenucleic acid molecules or parts thereof. The molecules hybridizing withthe nucleic acid molecules also comprise fragments, derivatives andallelic variants of the above-described nucleic acid molecules, whichcode for an enzyme usable according to the invention. Fragments heremean parts of nucleic acid molecules, which are long enough to encodethe enzyme described. Derivative means sequences of these molecules,which differ from the sequences of the above-described nucleic acidmolecules in one or more positions but which have a high degree ofhomology to these sequences. Homology here means a sequence identity ofat least 40%, in particular an identity of at least 60%, preferably over80% and particularly preferably over 90%, 95%, 97% or 99%, at thenucleic acid level. The encoded enzymes here have a sequence identity tothe amino acid sequences specified of at least 60%, preferably of atleast 80%, particularly preferably of at least 95%, very particularlypreferably at least 99%, at the amino acid level. The deviations heremay be the result of deletion, substitution, insertion or recombination.They may be naturally occurring variations, for example sequences fromother organisms, or else mutations which may occur naturally or byspecific mutagenesis (UV rays, X rays, chemical agents or others). Thevariants may also be synthetically produced sequences. These variantshave particular common characteristics such as, for example, enzymeactivity, active enzyme concentration, subunits, functional groups,immunological reactivity, conformation and/or physical properties suchas the migration behavior in gel electrophoresis, chromatographicbehavior, solubility, sedimentation coefficients, pH optimum,temperature optimum, spectroscopic properties, stability and/or others.

The invention furthermore also relates to the novel proteins with thesequence No. 2 and 4 and to a heterodimeric protein comprising thesequence No. 2 and sequence No. 4 and their at least 99% homologs.

SEQ ID NO: 1 depicts the 1644 bp nucleotide sequence for the largesubunit of the cobalamin-dependent mutase from DSM 18028.

SEQ ID NO: 2 depicts the 548 aa amino acid sequence of the large subunitof the cobalamin-dependent mutase from DSM 18028.

SEQ ID NO: 3 depicts 369 bp of the partial nucleotide sequence for thesmall subunit of the cobalamin-dependent mutase from DSM 18028.

SEQ ID NO: 4 depicts the 123 aa partial sequence of the subunit of thecobalamin-dependent mutase from DSM 18028.

SEQ ID NO: 5 and 6 depict the 562 and 135 aa, respectively, amino acidsequences of a cobalamin-dependent mutase from Xanthobacterautotrophicus Py2.

SEQ ID NO: 7 and 8 depict the 1689 and 408 bp, respectively, of thenucleotide sequence for the cobalamin-dependent mutases fromXanthobacter autotrophicus Py2.

SEQ ID NO: 9 and 10 depict the 563 and 135 aa, respectively, amino acidsequences of a cobalamin-dependent mutase from Rhodobacter sphaeroidesATCC 17029.

SEQ ID NO: 11 and 12 depict the 1692 and 408 bp, respectively, of thenucleotide sequence for the cobalamin-dependent mutases from Rhodobactersphaeroides ATCC 17029.

SEQ ID NO: 13 and 14 depict the 569 and 164 aa, respectively, amino acidsequences of a cobalamin-dependent mutase from Nocardoides sp. JS614.

SEQ ID NO: 15 and 16 depict the 1710 and 495 bp, respectively, of thenucleotide sequence for the cobalamin-dependent mutases from Nocardoidessp. JS614.

The 2-hydroxy-2-methyl carboxylic acids produced according to theinvention may be isolated by treating the culture medium (after removingundissolved components such as microbial cells) by previously disclosedmethods. Examples of such methods are, among others, concentration, ionexchange, distillation, electrodialysis, extraction and crystallization.The product may be isolated as salt or (after acidification) asprotonated 2-hydroxy-2-methyl carboxylic acid.

2-hydroxy-2-methyl carboxylic acids (or their corresponding salts) maybe dehydrated by a multiplicity of methods to give the correspondingunsaturated 2-methyl carboxylic acids. C2-C3-unsaturated isoalkenoicacids are produced by dehydrating the 2-hydroxy-2-methyl carboxylic acidproduced, using the known methods of the prior art. The2-hydroxy-2-methyl carboxylic acids may be dehydrated using metaloxides, metal hydroxides, ion exchange resins, alumina, silicon dioxide,amines, phosphines, alkali metal alkoxides and alkali metalcarboxylates. Reaction temperatures are usually between 160° C. and 250°C. Thus, for example, methacrylic acid is produced by dehydrating2-hydroxy isobutyric acid in the presence of NaOH at temperatures ofapprox. 185° C.

The methacrylic acid produced by this process and its homologs areappropriately applied in a whole number of industrial sectors, forexample as additives and in coatings. In contrast to the previouslyknown methods, the method combines the desired advantages of a lowtemperature process, the use of environmentally benign reactants andlower waste production.

The invention will be described in more detail below on the basis ofexemplary embodiments but is not intended to be limited thereto.

Material and Methods Microbial Enzyme Catalyst

Microbial cells of strain HCM-10 (DSM 18028), characterized by a3-hydroxy-carbonyl-CoA ester-producing and 3-hydroxy-carbonyl-CoAester-isomerizing activity, or the protein subunits with sequence No. 2and No. 4 isolated therefrom.

Growth of the Microbial Enzyme Catalysts

The microbial strain used for the production of 2-hydroxy-2-methylcarboxylic acids was isolated as described hereinbelow. Stock culturesare stored in 20% strength glycerol solution in liquid nitrogen.

Strain HCM-10 was concentrated from ground water on a basal medium(table 1) containing tert-butyl alcohol as sole carbon and energysource.

The strain belongs phylogenetically to the Rubrivivax-Leptothrix group.

TABLE 1 Basal medium (mg/L) NH₄Cl 761.4 KH₂PO₄ 340.25 K₂HPO₄ 435.45CaCl₂ × 6 H₂O 5.47 MgSO₄ × 7 H₂O 71.2 ZnSO₄ × 7 H₂O 0.44 MnSO₄ × H₂O0.615 CuSO₄ × 5 H₂O 0.785 CoCl₂ × 6 H₂O 0.2 Na₂MoO₄ × 2 H₂O 0.252 FeSO₄× 7 H₂O 4.98 Biotin 0.02 Folic acid 0.02 Pyridoxine-HCl 0.1 Thiamin-HCl0.05 Riboflavin 0.05 Nicotinic acid 0.05 DL-Ca-pantothenate 0.05p-aminobenzoic acid 0.05 Liponic acid 0.05 pH 7.0

Strain HCM-10 was grown aerobically under the following conditions(table 2) for assaying 3-hydroxy-carbonyl-CoA mutase activity.

TABLE 2 Temperature Time Strain Substrate Medium (° C.) (d) HCM-10tert-butyl alcohol Basal 25 7 (0.5 g/L) medium

The cells were used immediately after harvesting. Intact cells may beused without further pretreatment such as, for example,permeabilization. Moreover, the cells may be used in a permeabilizedform (for example by treatment with toluene, detergents or byfreeze-thaw cycles) in order to improve the rates of diffusion ofsubstances into the cells and out of the cells.

The concentration of 2-hydroxy isobutyric acid and 3-hydroxy butyricacid in the culture liquid or in the reaction mixture were determined bygas chromatography after acidic methanolysis, utilizing an FFAP and anFID detector.

EXAMPLE 1 Conversion of 3-hydroxy butyric acid to 2-hydroxy isobutyricacid by strain HCM-10

A suspension of 1 g (dry mass) of cells of strain HCM-10 in 100 ml ofbasal medium was introduced into 120 ml serum bottles. This suspensionwas admixed with 50 mg of 3-hydroxy butyric acid, and the suspension wasincubated on a rotary shaker at 30° C. After 0.3 h of aerobicincubation, the suspension was gassed with nitrogen and incubated withshaking at 30° C. for another 4.4 h. At various times, samples weretaken and the 2-hydroxy isobutyric acid content and 3-hydroxy butyricacid content in the cell-free supernatant were determined aftercentrifugation of the suspension. 2-hydroxy isobutyric acid was found tobe the sole product released in the anaerobic phase. In contrast,3-hydroxy butyric acid was evidently completely degraded in the aerobicinitial phase (FIG. 1). The yield of 2-hydroxy isobutyric acid was inthis case 5.1%, with approx. 80% of 3-hydroxy butyric acid remaining inthe reaction liquid.

EXAMPLE 2 Conversion of 3-hydroxy butyric acid to 2-hydroxy isobutyricacid by a crude extract of strain HCM-10

Cell-free crude extract of strain HCM-10 was prepared by disintegratingthe cells in a ball mill, and cell debris was subsequently removed bycentrifugation.

Cell-free crude extract at a concentration of 10 mg of protein in 5 mlof 50 mM potassium phosphate buffer (contains 1 mM MgCl₂ at pH 7.2) wasintroduced into sealable 10 ml glass vessels. To this extract were thenadded 0.01 mM coenzyme B12, 1 mM coenzymeA, 1 mM ATP and 4.25 mg of3-hydroxy butyric acid. The reaction liquid was gassed with nitrogen,the reaction vessel was sealed tightly and incubated with shaking at 30°C. for 2 h. The reaction products were analyzed as illustrated above.The yield of 2-hydroxy isobutyric acid was in this case 9%, with approx.88% of 3-hydroxy butyric acid remaining in the reaction liquid (FIG. 2).

EXAMPLE 3 Dehydration of 2-hydroxy isobutyric acid to methacrylate

A solution of 2-hydroxy isobutyric acid (1 mg/5 ml) produced accordingto the procedure carried out in example 2 was admixed with NaOH (0.06mg) with stirring. The solution was incubated with stirring and coolingat reflux under reduced pressure (300 torr) at 185-195° C. Furtheraliquots of 0.5 mg of 2-hydroxy isobutyric acid per 5 ml were addedevery hour over a period of 5 h, said aliquots additionally containing0.4 percent by weight of p-methoxyphenol in order to preventpolymerization of methacrylate. The reaction was stopped after 24 h ofincubation. The conversion of 2-hydroxy isobutyric acid to methacrylatewas 97%. Methacrylic acid was removed from the reaction mixture bydestillation.

1. A method for the enzymatic production of 2-hydroxy-2-methylcarboxylic acids from 3-hydroxy carboxylic acids, characterized in thata 3-hydroxy carboxylic acid is produced in and/or added to an aqueousreaction solution comprising a unit having 3-hydroxy-carboxylate-CoAmutase activity, which has both 3-hydroxy-carbonyl-CoA ester-producingand 3-hydroxy-carbonyl-CoA ester-isomerizing activity, and, afterincubation, the correspondingly converted 2-hydroxy-2-methyl carboxylicacid is isolated as acid or in the form of its salts.
 2. A method asclaimed in claim 1, characterized in that the unit having3-hydroxy-carboxylate-CoA mutase activity comprises an isolatedcobalamin-dependent mutase.
 3. A method as claimed in claim 1,characterized in that the unit having 3-hydroxy-carboxylate-CoA mutaseactivity comprises a 3-hydroxy-carbonyl-CoA ester-producing enzyme orenzyme system.
 4. A method as claimed in claim 1, characterized in thatthe unit having 3-hydroxy-carboxylate-CoA mutase activity is amicroorganism or a crude extract thereof.
 5. A method as claimed inclaim 1, characterized in that microorganisms producing or comprising acobalamin-dependent mutase, having 3-hydroxy-carboxylate-CoA mutaseactivity and having both 3-hydroxy-carbonyl-CoA ester-producing and3-hydroxy-carbonyl-CoA ester-isomerizing activity are used in aqueoussystems for converting 3-hydroxy carboxylic acids to the corresponding2-hydroxy-2-methyl carboxylic acid.
 6. A method as claimed in claim 1,characterized in that a) microorganisms with 3-hydroxy-carboxylate-CoAmutase activity, which have both 3-hydroxy-carbonyl-CoAthioester-producing activity and 3-hydroxy-carbonyl-CoAthioester-isomerizing activity, are cultured in an aqueous system withrenewable raw materials or waste products deriving from the consumptionof renewable raw materials as carbon and energy sources, wherebyintracellular 3-hydroxy-carboxylate-CoA thioesters are synthesized andconverted to the corresponding 2-hydroxy-2-methyl carboxylic acids, andb) the corresponding 2-hydroxy-2-methyl carboxylic acid is isolated asacid or in the form of its salts.
 7. A method as claimed in claim 1,characterized in that the reaction is carried out with the addition ofexternal 3-hydroxy carboxylic acid.
 8. A method as claimed in claim 1,characterized in that the microorganism is selected from the bacteriastrain HCM-10 DSM 18028, Xanthobacter autotrophicus Py2, Rhodobactersphaeroides (ATCC 17029) or Nocardioides sp. JS614.
 9. A method asclaimed in claim 1, characterized in that intact cells of themicroorganisms are used unchanged, permeabilized or fixed to a support.10. A method as claimed in claim 8, characterized in that cell extractsand/or the cobalamin-dependent mutase and, the other enzymes such as,CoA ester-synthesizing enzymes, after partial or complete isolation fromthe microorganisms are used, where appropriate in a purified form.
 11. Amethod as claimed in claim 10, characterized in that cell-free crudeextracts of the microorganisms are used.
 12. A method as claimed inclaim 1, characterized in that the proteins with the sequences SEQ IDNO: 2 and/or SEQ ID NO: 4, with the sequences SEQ ID NO: 5 and/or SEQ IDNO: 6, the proteins with the sequences SEQ ID NO: 9 and/or SEQ ID NO:10, or the proteins with the sequences SEQ ID NO: 13 and/or SEQ ID NO:14 are used and their at least 60% homologs.
 13. A method as claimed inclaim 1, characterized in that a sugar and/or an alcohol and/or anorganic acid and/or a hydrocarbon or their derivatives are used ascarbon and energy sources for enzymatic conversion during culturing. 14.A method as claimed in claim 13, characterized in that a substrate witha tert-butyl radical is used as carbon source and energy source forculturing.
 15. A method as claimed in claim 14, characterized in thattert-butyl alcohol is used as sole carbon source and energy source in abasal medium for culturing.
 16. A method as claimed in claim 1,characterized in that the enzymatic conversion of a sugar and/or analcohol and/or an organic acid and/or a hydrocarbon or their derivativesto the 3-hydroxy carboxylic acid and of the 3-hydroxy carboxylic acid tothe 2-hydroxy-2-methyl carboxylic acid is carried out in a single methodstep.
 17. A method as claimed in claim 1, characterized in that2-hydroxy isobutyric acid is produced with the addition of external3-hydroxy butyric acid.
 18. A method for the production ofC2-C3-unsaturated isoalkenoic acids, characterized in that a2-hydroxy-2-methyl carboxylic acid produced as claimed in claim 1 isdehydrated.
 19. A method for the production of methacrylic acid,characterized in that a 2-hydroxy isobutyric acid produced as claimed inclaim 1, is dehydrated.
 20. A microorganism strain HCM-10—DSM
 18028. 21.A nucleic acid molecule coding for an enzyme having the activity of acobalamin-dependent mutase, selected from the group consisting of a)nucleic acid molecules coding for a protein comprising the amino acidsequences indicated under Seq. No. 2 and/or Seq. No. 4; b) nucleic acidmolecules comprising the nucleotide sequence depicted under Seq. No. 1and/or Seq. No.
 3. 22. A nucleic acid molecule as claimed in claim 21,characterized in that it codes for a protein which, as an oligomericenzyme composed of two different subunits, comprises the amino acidsequences indicated under Seq. No. 2 and Seq. No.
 4. 23. A nucleic acidmolecule as claimed in claim 21, which is a DNA molecule.
 24. A nucleicacid molecule as claimed in claim 23, which is a cDNA or genomic DNA.25. A nucleic acid molecule as claimed in claim 21, which is an RNAmolecule.
 26. A protein having the activity of a cobalamin-dependentmutase encoded by a nucleic acid molecule as claimed in claim
 21. 27. Aprotein with the sequence No. 2 and its at least 99% homologs.
 28. Aprotein with the sequence No. 4 and its at least 99% homologs.
 29. Aprotein as heterodimeric enzyme comprising sequence No. 2 and sequenceNo. 4.